Acanthamoeba castellanii, an opportunistic free-living amoeba, causes severe infections including Acanthamoeba keratitis. This exploratory study evaluated whether three non-steroidal anti-inflammatory drugs (NSAIDs)—acetylsalicylic acid, ibuprofen, and diclofenac (100 µM)—modulate pathogenicity-related processes in A. castellanii and explored the involvement of a gp63-like protein during encystment and adhesion. Trophozoites were continuously exposed to each drug and analyzed for adhesion, migration on host-derived discontinuous brain micropatterns, encystment efficiency, and parasite-induced cytoskeletal remodeling in MDCK epithelial cells. In silico docking was performed to assess potential drug–protein interactions. Drug exposure reduced adhesion with maximal inhibition at 60 min. After 1 h, migration decreased by 49%, 64%, and 38%, and encystment was reduced by 50%, 85%, and up to 90%, respectively, in cultures treated with acetylsalicylic acid, ibuprofen, and diclofenac. Co-incubation with untreated trophozoites lowered actin fluorescence to approximately 50%, whereas drug-treated co-cultures preserved fluorescence near control levels. Colocalization analysis showed increased spatial overlap between gp63-like protein and F-actin in cysts (~40%) and migrating trophozoites (~20%) compared with non-stimulated forms (~3.8%). Collectively, these findings suggest that NSAID-sensitive pathways influence host interaction, migration, and encystment in A. castellanii and allow for the proposal of gp63-like protein as a putative molecular component of the NSAIDs sensitive pathways.

Activation of host inflammatory signaling pathways represents a critical determinant of tissue responses during Leishmania infection. Prostaglandin E₂ (PGE₂), synthesized by cyclooxygenase-2 (COX-2), has been implicated in the modulation of macrophage activation and disease pathology; however, the intracellular mechanisms regulating its production in specific host cell contexts remain incompletely defined. In the present study, we investigated the contribution of mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways to the regulation of the COX-2/PGE₂ axis during infection of J774A.1 macrophages with Leishmania mexicana promastigotes. Infection induced rapid and sustained ERK1/2 activation together with increased COX-2 expression and PGE₂ synthesis. In contrast, p38 MAPK activation was delayed and transient, declining rapidly compared with the sustained ERK1/2 response, while JNK phosphorylation remained minimal under the experimental conditions evaluated. Pharmacological inhibition of NF-κB signaling significantly reduced inflammatory mediator production without affecting early parasite internalization. In a BALB/c model of cutaneous leishmaniasis, local administration of the NF-κB inhibitor BAY11-7082 was associated with decreased lesion progression, suggesting that modulation of host inflammatory signaling may influence tissue pathology during infection. In axenic cultures, the compound produced a delayed reduction in parasite proliferation, indicating that potential direct antiparasitic effects may be limited under the experimental conditions evaluated. Overall, these findings support a role for coordinated MAPK and NF-κB activation in the regulation of COX-2-dependent lipid mediator production during L. mexicana infection and highlight the relevance of context-dependent host inflammatory signaling as a complementary factor influencing disease progression.